1. Field of the Invention
Protein binding assays or immunoassays have been the subject of thorough investigation and commercialization. The ability to specifically determine a drug or other compound of interest, particularly physiological interest, which is present in extremely low concentration, usually less than micrograms per milliliter, has opened up new opportunities in clinical laboratories. The ability to monitor therapeutic drug administration or drug addiction, to rapidly and efficiently determine diseased states, and to monitor the condition of a patient during times of stress, has provided significant opportunities for improvement of health care.
The assays in the clinical laboratory frequently require not only high sensitivity but accuracy over a relatively narrow range. Therefore, new techniques are being developed which recognize these requirements by providing for greater sensitivity, reduced response to non-specific effects, and easier and simplier protocols. In addition, many antigens can be obtained only in impure form or in pure form at elevated costs. Therefore, assays must accommodate the possibility that the antigen will be impure. Parallel to this situation is that most antibodies which are obtained by antigenic injection will have less than about 30 weight %, or frequently less than about 20 weight % of the total protein as the antibody of interest. When preparing reagents which involve reactions with the antibody composition, the presence of the large amount of contaminant must be taken into account.
Other considerations in developing an assay include the necessity for and number of incubations, the period required for the incubation, the period required for the measurement, the sensitivity of the measurement to extraneous factors, the stability of the reagents, the formulation of the reagents, and the like.
2. Description of the Prior Art
U.S. Pat. No. 3,996,345 describes the employment of a chromophore pair-fluorescer and quencher-, where the members of the pair are bonded to different members of a specific binding pair, so that the amount of fluorescer and quencher which come within an interacting distance is dependent upon the amount of analyte in the medium. U.S. Pat. No. 3,935,074 describes an immunoassay dependent upon the inability of two antibodies to simultaneously bind to a reagent having at least two determinant sites. Co-pending application Ser. No. 893,650, filed Apr. 5, 1978, now U.S. Pat. No. 4,233,402, teaches the concept in immunoassays of bringing together two enzymes, whose substrates or products are in some ways related to the production of a detectible signal, where the juxtaposition of the enzymes is related to the amount of analyte in the medium. Co-pending application Ser. No. 815,632, filed July 14, 1977, now U.S. Pat. No. 4,208,479, teaches the employment of a macromolecular modifier of a label bound to an antibody, where the modifier is inhibited from approaching the label when the labeled antibody is bound to antigen. Co-pending application Ser. No. 815,487, filed July 14, 1977, now U.S. Pat. No. 4,233,401, discloses an enzyme immunoassay where ligand is labeled with enzyme and an enzyme inhibitor is inhibited from approaching the enzyme, when antibody is bound to the ligand. Co-pending application Ser. No. 964,099, filed Nov. 24, 1978, discloses the use of macromolecular particles to provide discrimination between a label bound to the particle and a label free in the solution, where the amount of label bound to the particle is related to the amount of analyte in the medium, and the observed detectible signal is dependent upon the distribution of the label between the particle and the medium. U.S. Pat. No. 3,817,837 describes a homogeneous enzyme immunoassay.